AU2021204727A1 - Porcine g-csf variants and their uses - Google Patents

Porcine g-csf variants and their uses Download PDF

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AU2021204727A1
AU2021204727A1 AU2021204727A AU2021204727A AU2021204727A1 AU 2021204727 A1 AU2021204727 A1 AU 2021204727A1 AU 2021204727 A AU2021204727 A AU 2021204727A AU 2021204727 A AU2021204727 A AU 2021204727A AU 2021204727 A1 AU2021204727 A1 AU 2021204727A1
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Peter Connor Canning
Nickolas KNUDSEN
Md Harunur RASHID
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Elanco US Inc
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Abstract

The present invention relates to variants of porcine granulocyte colony stimulating factor (pG CSF). The pG-CSF variants are useful in treating preventing or reducing the incidence of bacterial infections in swine. Methods of treating swine are disclosed.

Description

PORCINE G-CSF VARIANTS AND THEIR USES
The present application is a divisional application from Australian Patent Application No. 2018348138, the entire disclosure of which is incorporated herein by reference.
This application claims the benefit of priority to US Provisional patent application serial number 62/570,877, filed October 11, 2017, and which is incorporated herein by reference in its entirety. The present invention relates to variant porcine granulocyte colony stimulating factor (pG-CSF) polypeptides containing a synthetic amino acid. The synthetic amino acid is modified by attachment of a poly(ethylene glycol) (PEG) molecule. The PEGylated pG-CSF variant is used to treat bacterial infections in a porcine. When the porcine is a periparturient sow, the bacterial infection may be mastitis, metritis, and agalactia (MMA) syndrome. Reducing bacterial infections in pregnant sows improves piglet survival. The economic impact of infectious diseases in food animal production is well documented. Infectious diseases reduce profits, increase production costs, and endanger food products, as well as affect the performance, health, and welfare of the animal. Diseases can cause morbidity and mortality of newborn, young (e.g., replacement stock) or adult animals, resulting in devastating effects on food animal production. In porcines, one such disease may be mastitis, metritis, and agalactia (MMA) syndrome. Mastitis is a bacterial infection of the udder. Only one or two glands may be affected, or the infection could spread to multiple glands. Metritis is a bacterial infection of the urogenital tract, sometimes presented as vulval discharges. Agalactica is a reduction in, or the total loss of, milk production by a sow. MMA syndrome can be highly variable and may not present all the above symptoms. Thus, MMA syndrome can be difficult to detect and diagnose, and it may not be detected until a nursing piglet shows signs of hunger, weight loss, or even death. Because MMA syndrome is difficult to detect, a prophylactic treatment is preferable. Use of antibiotics, particularly shared class antibiotics, is discouraged in food-producing animals, so a non-antibiotic therapy is preferable in the treatment of porcines. A cytokine such as pG-CSF could increase neutrophil numbers in an animal, thus priming the innate immune system to respond quickly to a bacterial infection. Modifying a pG-CSF variant with PEG could prolong the pharmokinetics and stability of the cytokine, thus potentiating its effects. A PEGylated bovine G-CSF can be used to treat mastitis in dairy cattle (W02010/011735). PEGylated human G-CSF has also been described (W02000/044785). A
PEGylated wild type porcine G-CSF has been proposed for the treatment of respiratory infections, such as viral infections (W02005/025593). As disclosed in various aspects herein, a PEGylated pG-CSF variant elevates porcine blood neutrophil numbers, reduces the incidence of MMA syndrome in periparturient sows, and/or improves piglet survival. The present invention provides porcine granulocyte colony stimulating factor (pG CSF) variants having a consensus sequence of: X 1PLSPASSLPQSFLLKX 2LEQVRKIQADGAELQERLCATHKLCX 3PQELVLLGHSLGLP QASLSSCSSQALQLTGCLNQLHGGLVLYQGLLQALAGISPELAPALDILQLDVTDLAT NIWLQX 4EDLRX 4APASLPTQGTVPTFTSAFQRRAGGVLVVSQLQSFLELAYRVLRYL AEP (SEQ ID NO: 13). The variable X1 can be the dipeptide methionine alanine as in SEQ ID NOs: 2 and 9, the dipeptide norleucine alanine as in SEQ ID NOs:7 and 10, alanine as in SEQ ID NOs:4 and 11, or absent as in SEQ ID NOs: 8 and 12. The variable X 2 can be cysteine as in SEQ ID NOs: 2, 4, 7, and 8, or X 2 can be seine as in SEQ ID NOs: 9, 10, 11, and 12. The variable X3 can be a synthetic amino acid. The synthetic amino acid can be present at position 43 (position relative to the mature wild-type pG-CSF as given in SEQ ID NO: 3) as shown in SEQ ID NO: 13. The synthetic amino acid can be para-acetyl phenylalanine (pAF). The variable X4 can be methionine as in SEQ ID NOs:2, 4, 8, 9, 11, and 12, or X4 can be norleucine as in SEQ ID NOs:7 and 10. The pAF synthetic amino acid can be covalently attached to a poly(ethylene glycol) (PEG) molecule. The PEG can have a molecular weight of about 20 kD to about 50 kD, or a molecular weight of about 30 kD. Preferably, the PEG is a linear PEG molecule. The present invention provides a porcine granulocyte colony stimulating factor (pG CSF) variant having a sequence of: MAPLSPASSLPQSFLLKCLEQVRKIQADGAELQERLCATHKLC[pAF]PQELVLLGHSL GLPQASLSSCSSQALQLTGCLNQLHGGLVLYQGLLQALAGISPELAPALDILQLDVTD LATNIWLQMEDLRMAPASLPTQGTVPTFTSAFQRRAGGVLVVSQLQSFLELAYRVLR YLAEP (SEQ ID NO: 2), wherein a para-acetyl phenylalanine (pAF) synthetic amino acid present at position 43 is covalently attached to a 30 kD linear PEG molecule. The present invention provides a pharmaceutical composition comprising any of the pG-CSF variants described herein, and at least one pharmaceutically acceptable carrier, diluent, or excipient.
The present invention provides a method for treating a bacterial infection in a porcine comprising administering a therapeutically effective amount of any of the pG-CSF variants described herein to the porcine in need thereof. The bacterial infection may be mastitis, metritis and agalactia (MMA) syndrome. The porcine in need of treatment may be a periparturient sow. The therapeutically effective amount of a pG-CSF variant may be about 10 - 100 pg/kg animal weight, or about 30 - 50 pg/kg animal weight, or about 40 pg/kg animal weight. Administration of any of the pG-CSF variants described herein may occur at least once 7 days or less prior to farrowing (i.e. on or after day 107 of gestation), or at farrowing. In some aspects, the method of treating a bacterial infection in a porcine may comprise a second administration of any of the pG-CSF variants described herein within 14 days or less after farrowing (i.e., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or within 14 days after farrowing). The present invention provides a method for stimulating innate immune response in a porcine comprising administering a therapeutically effective amount of any of the pG-CSF variants described herein to the porcine in need thereof. The porcine in need of stimulation may be a periparturient sow. In other aspects the porcine in need of stimulation may be a porcine with a compromised or weakened immune system. In further aspects the porcine in need of stimulation may be a porcine that has or is at risk of developing an infection including, for example a bacterial infection or a viral infection. In some aspects, the method can stimulate production of cytokines (e.g., interferons (IFNs), tumor necrosis factors (TNFs), colony stimulating factors (CSFs), and/or interleukins (ILs)), and/or activate or increase levels of immune cells such as dendritic cells (DCs), lymphocytes (e.g., B cells, T cells, and natural killer (NK) cells), and myelocytes (e.g., Mast cells, myeloblasts (e.g., basophils, eosinophils, neutrophils, monocytes, and macrophages)). In some embodiments of these aspects, the method can stimulate or enhance neutrophil antibacterial function by, for example, increasing neutrophil myeloperoxidase-hydrogen peroxide-halide mediated antibacterial function. In methods that stimulate the innate immune response, the therapeutically effective amount of a pG-CSF variant may be about 10 - 100 pg/kg animal weight, or about 30 - 50 pg/kg animal weight, or about 40 pg/kg animal weight. Administration of any of the pG-CSF variants described herein may occur at least once 7 days or less prior to farrowing (i.e. on or after day 107 of gestation), or at farrowing. In some aspects, the method of treating a bacterial infection in a porcine may comprise a second administration of any of the pG-CSF variants described herein within 14 days or less after farrowing (i.e., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or within 14 days after farrowing). The present invention provides a method for reducing piglet mortality comprising administering a therapeutically effective amount of any of the pG-CSF variants described herein to a periparturient sow. The therapeutically effective amount of a pG-CSF variant may be about 10-100 jg/kg animal weight, or about 30 - 50 pg/kg animal weight, or about 40 pig/kg animal weight. The administration of any of the pG-CSF variants described herein may occur at least once 7 days or less prior to farrowing (i.e. on or after day 107 of gestation), or at farrowing. In some aspects, the method of reducing piglet mortality may comprise a second administration to the periparturient sow of any of the pG-CSF variants described herein within 14 days or less after farrowing (i.e., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or within 14 days after farrowing). The present invention provides for use of any of the pG-CSF variants described herein in the manufacture of a medicament for a bacterial infection in a porcine. The bacterial infection may be MMA syndrome. The porcine may be a periparturient sow. The present invention provides for use of any of the pG-CSF variants described herein in the manufacture of a medicament for reducing piglet mortality. The present invention provides any of the pG-CSF variants described herein for use in therapy. The therapy may be the treatment of a syndrome of bacterial infection in a porcine. The bacterial infection may be MMA syndrome. The porcine may be a periparturient sow. The therapy may be the reduction of piglet mortality. The pG-CSF variant may be administered to a periparturient sow at about 10 - 100 jg/kg animal weight, or about 30 - 50 jig/kg animal weight, or about 40 jg/kg animal weight. The pG-CSF variant may be administered at least once 7 days or less prior to farrowing (i.e. on or after day 107 of gestation), or at farrowing. A second administration may be given to the periparturient sow 14 days or less after farrowing. A "bacterial infection" is the growth of one or more bacterial species on or within the skin, mucus membranes, glands, eyes, ears, urogenital tract, digestive tract, lungs, blood or organs of an animal. The bacteria may be a species classified within the genera of Achromobacter, Actinobacillus, Actinomyces, Bacillus, Bordatella, Brucella, Clostridium, Corynebacterium, Erysipelothrix, Escherichia, Haemophilus, Klebsiella, Leptospira, Listeria,
Mycoplasma, Pasteurella,Proteus, Pseudomonas, Salmonella, Sphaerophorus, Staphylococcus, Streptococcus, or Vibrio. The term "porcine" as used herein, refers to a pig, especially the domestic pig (Sus scrofa domesticus or Sus domesticus) and can include miniature pigs as well as those breeds raised for meat production. By "pig", "swine" or "porcine" is meant to include all pig breeds. The periparturient sow is defined as the pregnant female porcine within the last weeks of gestation through the first few weeks post-farrowing. Farrowing, or giving birth, typically occurs at about 114 days of gestation. A periparturient sow could be a pregnant female porcine from about 100 days of gestation to about 14 days post-farrowing. A piglet is a porcine from birth to weaning at about 3 weeks of age. A "synthetic amino acid" refers to an amino acid that is not one of the 20 common amino acids or pyrrolysine or selenocysteine. Examples of such synthetic amino acids include, but are not limited to, para-acetyl phenylalanine (pAF), acetylglucosaminyl-L-serine, and N- acetylglucosaminyl-L-threonine. Some synthetic amino acids and their incorporation into polypeptides and subsequent modification are described in W02010/011735 and in W02005/074650. As used herein, the terms"treating", "to treat", or "treatment", include restraining, slowing, stopping, reducing, ameliorating, or reversing the progression or severity of a symptom, disorder, condition, or disease. In some aspects the disclosure provides methods that are specific to prophylactic treatment of a symptom, disorder, condition, or disease that is not observed or detected in an animal which may be at risk of developing one or more such symptom, disorder, condition, or disease. In some aspects, a treatment will be applied therapeutically. The term "effective amount" as used refers to that amount of the pG-CSF variant being administered which will have the desired effect, such as preventing, treating, or reducing a bacterial infection in a porcine. When the porcine is a periparturient sow, treating bacterial infections in the sow can improve survival of piglets. The effective amount may vary with factors such as the weight of the sow. By "administering" is meant the injection of a therapeutically effective amount of the compounds and compositions containing said compounds disclosed. For example without limitation, administration can be intramuscular (i.m) or subcutaneous (s.c.).
The term "about" will be understood by persons of ordinary skill in the art and will vary to some extent depending on the context in which it is used. As used herein, "about" is meant to encompass variations of ±10%, ±5%, or ±1% (i.e., ±10%, ±9%,±8%, ±7%,±6%, ±5%, ±4%, ±3%, ±2%, or ±1%). The pG-CSF variants of the present invention may readily be produced in a variety of cells including mammalian cells, bacterial cells such as E. coli, Bacillus subtilis, or Pseudomonasfluorescence,and/or in fungal or yeast cells. The host cells can be cultured using techniques well known in the art. The vectors containing the polynucleotide sequences of interest (e.g., the variants of pG-CSF and expression control sequences) can be transferred into the host cell by well-known methods, which vary depending on the type of cellular host. For example, the calcium chloride transformation method is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment or electroporation may be used for other eukaryotic host cells. Various methods of protein purification may be employed and such methods are known in the art and described, for example, in Deutscher, Methods in Enzymology 182: 83-89 (1990) and Scopes, Protein Purification:Principlesand Practice, 3rd Edition, Springer, NY (1994). The PEGylated pG-CSF variants can be formulated according to known methods to prepare pharmaceutically useful compositions. In some aspects a formulation is a stable lyophilized product that is reconstituted with an appropriate diluent or an aqueous solution of high purity with optional pharmaceutically acceptable carriers, preservatives, excipients or stabilizers (see Remington, The Science and Practiceof Pharmacy, 19th ed., Gennaro, ed., Mack Publishing Co., Easton, PA 1995). The PEGylated pG-CSF variant may be formulated with a pharmaceutically acceptable buffer, and the pH adjusted to provide acceptable stability, and a pH acceptable for administration. In some non-limiting examples, a pH acceptable for administration may be in a range from about 5 to about 8 (i.e., about 5, about 6, about 7, or about 8). Moreover, the PEGylated pG-CSF compositions of the present invention may be placed into a container such as a vial, a cartridge, a pen delivery device, a syringe, intravenous administration tubing or an intravenous administration bag. The following experimental examples are illustrative of pG-CSF variants, and their use in treating bacterial infections in swine, such as MMA syndrome in a periparturient sow. Reducing the incidence of bacterial infections in the sow also reduces piglet mortality. It will be appreciated that other embodiments and uses will be apparent to those skilled in the art and that the invention is not limited to these specific illustrative examples or preferred embodiments.
EXAMPLE 1
Different variants of the porcine granulocyte colony stimulating factor (pG-CSF) cDNA (GenBank Accession number U68481.1) are generated by introducing a TAG stop codon in the selected positions by site-directed PCR mutagenesis. Also, the portion of the cDNA encoding the signal sequence is replaced by a single methionine codon (i.e. ATG). For example, a cDNA encoding wild type mature pG-CSF with the altered signal sequence could be: 1 atggcccctc tcagccctgc cagctccctg cccagagct tcctgctcaa gtgcttagag 61 caagtgagga aaatccaggc tgatggcgcc gagctgcagg agaggctgtg tgccacccac 121 aagctgtgcc acccccagga gctggtgctg ctcgggcact ctctgggcct cccccaggct 181 tcCCtgagCa gctgctCcag ccaggcCCtg cagctgactg gctgcctgaa ccaactgcat 241 ggcggcctcg tcctctacca gggcctcctg caggccctgg cgggcatctc cccagagctg 301 gcccccgccc tggacatact gcagctggat gtcaccgact tagccaccaa catctggctg 361 cagatggaag acctgaggat ggccccggcc tcgcttccca cccagggcac cgtgccgacc 421 ttcacctcgg ccttccagcg ccgggcagga ggggtcctgg ttgtctccca gctgcagagc 481 ttcctggagc tggcgtaccg tgtcctgcgc tacCtCgccg agccctga
(SEQ ID NO: 5). This variant would encode a polypeptide of: 1 MAPLSPASSL PQSFLLKCLE QVRKIQADGA ELQERLCATH KLCHPQELVL LGHSLGLPQA 61 SLSSCSSQAL QLTGCLNQLH GGLVLYQGLL QALAGISPEL APALDILQLD VTDLATNIWL 121 QMEDLRMAPA SLPTQGTVPT FTSAFQRRAG GVLVVSQLQS FLELAYRVLR YLAEP
(SEQ ID NO: 1). If the TAG stop codon is desired to replace the H43 residue, the cDNA sequence could be: 1 atggcccctc tcagccctgc cagctccctg cccagagct tcctgctcaa gtgcttagag 61 caagtgagga aaatccaggc tgatggcgcc gagctgcagg agaggctgtg tgccacccac 121 aagctgtgct agccccagga gctggtgctg ctcgggcact ctctgggcct cccccaggct 181 tccctgagca gctgctccag ccaggccctg cagctgactg gctgcctgaa ccaactgcat 241 ggcggcctcg tcctctacca gggcctcctg caggccctgg cgggcatctc cccagagctg 301 gcccccgccc tggacatact gcagctggat gtcaccgact tagccaccaa catctggctg 361 cagatggaag acctgaggat ggccccggcc tcgcttccca cccagggcac cgtgccgacc 421 ttcacctcgg ccttccagcg ccgggcagga ggggtcctgg ttgtctccca gctgcagagc 481 ttcctggagc tggcgtaccg tgtcctgcgc tacctcgccg agccctga
(SEQ ID NO: 6). The resulting polypeptide would be: 1 MAPLSPASSL PQSFLLKCLE QVRKIQADGA ELQERLCATH KLCxPQELVL LGHSLGLPQA 61 SLSSCSSQAL QLTGCLNQLH GGLVLYQGLL QALAGISPEL APALDILQLD VTDLATNIWL 121 QMEDLRMAPA SLPTQGTVPT FTSAFQRRAG GVLVVSQLQS FLELAYRVLR YLAEP
(SEQ ID NO: 2), where the "x" indicates the H43 residue replaced with a synthetic amino acid, as described below.
Plasmids encoding the variants are transformed into E coli cells containing the expanded genetic code system components for incorporation of the synthetic amino acid para acetyl phenylalanine (pAF). Transformed cells are grown in media supplemented with pAF and induced to express pG-CSF with pAF incorporated into the sites indicated. The expression system has been described, for example, in WO 2010/011735 (incorporated herein by reference), and is generally known in the art. Expression of the transfected cDNA variants is induced with arabinose, the cells are harvested, and the target pG-CSF pAF site variants are isolated and purified by reverse phase high-pressure liquid chromatography (RP-HPLC). An activated 30 kD linear aminooxy-PEG is site-specifically conjugated to the incorporated pAF. PEG-pG-CSF conjugates are purified from excess PEG and unconjugated pG-CSF variants by chromatography.
EXAMPLE 2 The in vitro biological activity of PEGylated pG-CSF (PEG-pG-CSF) variants is measured by the ability of the variants to induce proliferation of M-NSF-60 cells (ATCC CRL-1838). The concentration of the variants able to effect 50% of maximal proliferation (EC 5 0) is determined by comparison to a standard curve generated with wild type (WT) pG CSF. Based on the results of expression as presented in Example 1 and the biological assays described here, PEG-pG-CSF variants are selected for further study. The in vivo activity of the selected candidate PEG-pG-CSF variants is tested in a rodent model. Sprague Dawley rats (3/group) are treated with 0.25 mg/kg body weight with a PEG-pG-CSF variant. Blood samples are taken at 0 (pre-dosing), 1, 3, 6, 24, 48, 56, 72, 96, 144, 192, and 264 hours for pharmokinetic (PK) analysis, and samples are taken at 24, 48, 72, and 96 hours for a complete blood count (CBC analysis). The primary measurement in the CBC analysis is the number of neutrophils present. A H43 variant stimulates a higher level of neutrophil development than other variants tested. All variants have similar PK profiles.
EXAMPLE 3 The PEG-pG-CSF H43pAF is prepared as follows. As in Example 1, expression of the transfected cDNA variants is induced with arabinose, the cells are harvested, and the pG-CSF H43pAF site variant is isolated, denatured and refolded, and purified by cation exchange liquid chromatography (CEX), using CAPTO Adhere Impres (GE Healthcare Lifesciences).
Briefly, the unpegylated pG-CSF H43pAF variant is loaded onto the column to a concentration of 1-5 mg/mL resin. The column is washed with five column volumes (CV) 30 mM sodium acetate at pH 4.5. Elution of the pG-CSF H43pAF variant is with a linear gradient of elution buffer (30 mM sodium acetate, 0.5 M NaCl, Ph 4.5), by washing with 0 100% elution buffer over 20 CV. Based on mass spectroscopy (MS) analysis of pG-CSF H43pAF, the isolated peptides include a main peak represented by SEQ ID NO: 2 and several different contaminants. The contaminants include loss of the N-terminal methione (SEQ ID NO: 4), loss of both the N terminal methionine and alanine (SEQ ID NO: 8), and substitution of norleucine for the N terminal methionine (SEQ ID NO: 7). Norleucine is known to be misincorporated instead of the amino acid methionine in high density fermentation with E. coli. Norleucine incorporation is reduced by using one or more of the following steps: feeding the fermentation solutions with methionine; fermenting with complex media instead of defined media (the complex media has one or more non-defined components in it including but limited to glycerol, salts, amino acids, vitamins, yeast extracts, plant and animal hydrolysates, peptones, and tryptones); and/or lowering the temperature of the fermentation reaction mixture post induction. The pG-CSF H43pAF variant is taken from the cation exchange chromatography pool after using Capto SP Impres chromatography and buffer exchanged into 30mM sodium acetate, 4% sucrose, pH 4.0 using a lOkDa MWCO tangential flow filtration cassette. The pG-CSF H43pAF variant is then concentrated to about 8.0 mg/mL using an Amicon Ultra centrifugal filter according to manufacturer's instructions. Once concentrated, 30K linear PEG (PEG can be purchased commercially from NOF America Corporation or EMD Merck, for example) is added in a 6:1 molar ratio of PEG to pG-CSF H43pAF variant. The PEG /
pG-CSF variant mixture is then incubated at about 28°C for at least 21 hours. This method results in >98% of the pG-CSF variant being conjugated with PEG. The pegylated variant can then be purified by CEX as above. When tested in the M-NSF-60 cell bioassay (Example 2), the PEG-pG-CSF H43pAF variant has an EC5 0 of at least 0.40 ng/mL, demonstrating good binding and potency characteristics. Samples are frozen and thawed over five cycles by freezing at 0°C in 1.5 mL tubes and thawing in a room temperature water bath. No significant impact is observed for the high molecular weight (HMW) protein profile over five cycles of freeze-thawing, demonstrating the stability of the variant in solution. Two additional pG-CSF H43pAF variants are generated to attempt to improve refolding efficiency and thermostability of the variant at 50 °C. Cysteine 17 is changed to either alanine (C17A) or to seine (C17S, SEQ ID NOs: 9-12). These mutations do not improve refolding yield, but C17A has decreased thermostability. PEG-pG-CSF H43pAF/C17S does have a slightly improved EC5 0 of 0.26 ng/mL.
EXAMPLE 4 The PEG-pG-CSF H43pAF variant is administered to sows to characterize changes in blood neutrophils. Six sows of 1.5 - 5 years of age and an average body weight of 269.7 kg
are given 40fg/kg of the PEG-pG-CSF H43pAF variant by intramuscular injection on the
side of the neck. The PEG-pG-CSF H43pAF variant is suspended in 30mM sodium citrate, 250mM arginine, pH 6.0 at a concentration of 8.2 mg/mL. Animals do not receive any concomitant medication following initiation of treatment. No adverse events are observed. Blood is taken on day 0 prior to dosing and on days 2, 7, 10, 14, 17 and 21 post dosing. Neutrophil counts are determined for each sow and a mean for the treatment is determined for each day. Treatment with a single dose of the PEG-pG-CSF H43pAF variant results in measurable increases in blood neutrophil counts over a three-week period (Table 1). Additional doses would be expected to stimulate maintenance of the higher neutrophil levels.
Table 1. Effect of PEG-pG-CSF H43pAF variant on mean daily blood neutrophil counts. Day 0 2 7 10 14 17 21 Neutrophils (1000/tL) 4.65 43.68 25.08 30.1 18.53 15.14 9.83
EXAMPLE 5 The PEG-pG-CSF H43pAF variant is administered to periparturient sows to characterize the effect on mastitis, metritis, and agalactia (MMA) syndrome and on piglet survival. Sows at 95 - 100 days of gestation are placed into farrowing crates and hygienic husbandry practices are followed until day 107 of gestation for each sow. On day 107, blood is collected and then the sows (25 per group) are treated either with 40 g/kg of the PEG-pG CSF H43pAF variant as in Example 4 or with a sodium chloride solution as a negative control. The sows are then placed in unhygienic conditions to stimulate development of MMA. The unhygienic conditions include placing a mat on the grated floor of the farrowing crate to allow bedding and waste material to accumulate. Also, a mixture of water, feces, and pine sawdust (2:1:1) is used to contaminate the crates. No oxytocic or corticosteroid drugs are given to the sows. Clinical observations and rectal temperatures are collected on each sow once daily in the morning beginning on day 107 of gestation and continuing until a diagnosis of MMA at which time the sow has all samples collected and is then removed from the study, given treatment, and has hygienic crate conditions returned. Samples collected include blood, rectal temperature, and swabs of infected glands. Farrowing typically occurs on day 114 of gestation. Piglets are weighed and tagged within 12 hours of birth. Clinical observations of the piglets are made twice daily, and weights are also measured on days 3, 7, and 21 after birth. Using the per protocol definition of disease, little difference was observed between the two groups. However, when vulvar discharge was removed from the definition of disease, the incidence of disease was reduced by more than 50% in the PEG-pG-CSF treated group. The treated group also had a greater number of piglets weaned compared to control group (Table 2).
Table 2. Effect of PEG-pG-CSF on MMA incidence and piglet survival. Variable Control PEG-pG-CSF P value Sows (number/group) MMA 36% (9/25) 32%(8/35) 1.0000 MMA excluding vulvar discharge 28% (7/25) 12%(3/25) 0.2890 Piglets (Std. Error of Mean) Number born/litter 13.3 (0.78) 13.1 (0.72) 0.8567 Number weaned/litter 8.3 (0.61) 9.4 (0.61) 0.2056 Percent survival 65.3 (4.5) 72.4 (4.3) 0.2590 Range survival/litter 0 - 100% 38 - 100% n/a Weaning weight 5.9 (0.21) 5.7 (0.19) 0.5944
SEQUENCE LISTING
<210> 1 <211> 175 <212> PRT <213> Artificial Sequence
<220> <223> Artificial
<400> i
Met Ala Pro Leu Ser Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu 1 5 10 15
Lys Cys Leu Glu Gln Val Arg Lys Ile Gln Ala Asp Gly Ala Glu Leu 20 25 30
Gln Glu Arg Leu Cys Ala Thr His Lys Leu Cys His Pro Gln Glu Leu 35 40 45
Val Leu Leu Gly His Ser Leu Gly Leu Pro Gln Ala Ser Leu Ser Ser 50 55 60
Cys Ser Ser Gln Ala Leu Gln Leu Thr Gly Cys Leu Asn Gln Leu His 65 70 75 80
Gly Gly Leu Val Leu Tyr Gln Gly Leu Leu Gln Ala Leu Ala Gly Ile 85 90 95
Ser Pro Glu Leu Ala Pro Ala Leu Asp Ile Leu Gln Leu Asp Val Thr 100 105 110
Asp Leu Ala Thr Asn Ile Trp Leu Gln Met Glu Asp Leu Arg Met Ala 115 120 125
Pro Ala Ser Leu Pro Thr Gln Gly Thr Val Pro Thr Phe Thr Ser Ala 130 135 140
Phe Gln Arg Arg Ala Gly Gly Val Leu Val Val Ser Gln Leu Gln Ser 145 150 155 160
Phe Leu Glu Leu Ala Tyr Arg Val Leu Arg Tyr Leu Ala Glu Pro 165 170 175
<210> 2 <211> 175 <212> PRT <213> Artificial Sequence
<220> <223> Artificial
<220>
<221> MISCFEATURE <222> (44)..(44) <223> Xaa is pAF
<400> 2
Met Ala Pro Leu Ser Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu 1 5 10 15
Lys Cys Leu Glu Gln Val Arg Lys Ile Gln Ala Asp Gly Ala Glu Leu 20 25 30
Gln Glu Arg Leu Cys Ala Thr His Lys Leu Cys Xaa Pro Gln Glu Leu 35 40 45
Val Leu Leu Gly His Ser Leu Gly Leu Pro Gln Ala Ser Leu Ser Ser 50 55 60
Cys Ser Ser Gln Ala Leu Gln Leu Thr Gly Cys Leu Asn Gln Leu His 65 70 75 80
Gly Gly Leu Val Leu Tyr Gln Gly Leu Leu Gln Ala Leu Ala Gly Ile 85 90 95
Ser Pro Glu Leu Ala Pro Ala Leu Asp Ile Leu Gln Leu Asp Val Thr 100 105 110
Asp Leu Ala Thr Asn Ile Trp Leu Gln Met Glu Asp Leu Arg Met Ala 115 120 125
Pro Ala Ser Leu Pro Thr Gln Gly Thr Val Pro Thr Phe Thr Ser Ala 130 135 140
Phe Gln Arg Arg Ala Gly Gly Val Leu Val Val Ser Gln Leu Gln Ser 145 150 155 160
Phe Leu Glu Leu Ala Tyr Arg Val Leu Arg Tyr Leu Ala Glu Pro 165 170 175
<210> 3 <211> 174 <212> PRT <213> Artificial Sequence
<220> <223> Natural
<400> 3
Ala Pro Leu Ser Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys 1 5 10 15
Cys Leu Glu Gln Val Arg Lys Ile Gln Ala Asp Gly Ala Glu Leu Gln 20 25 30
Glu Arg Leu Cys Ala Thr His Lys Leu Cys His Pro Gin Glu Leu Val 35 40 45
Leu Leu Gly His Ser Leu Gly Leu Pro Gin Ala Ser Leu Ser Ser Cys 50 55 60
Ser Ser Gin Ala Leu Gin Leu Thr Gly Cys Leu Asn Gin Leu His Gly 65 70 75 80
Gly Leu Val Leu Tyr Gin Gly Leu Leu Gin Ala Leu Ala Gly Ile Ser 85 90 95
Pro Glu Leu Ala Pro Ala Leu Asp Ile Leu Gin Leu Asp Val Thr Asp 100 105 110
Leu Ala Thr Asn Ile Trp Leu Gin Met Glu Asp Leu Arg Met Ala Pro 115 120 125
Ala Ser Leu Pro Thr Gin Gly Thr Val Pro Thr Phe Thr Ser Ala Phe 130 135 140
Gin Arg Arg Ala Gly Gly Val Leu Val Val Ser Gin Leu Gin Ser Phe 145 150 155 160
Leu Glu Leu Ala Tyr Arg Val Leu Arg Tyr Leu Ala Glu Pro 165 170
<210> 4 <211> 174 <212> PRT <213> Artificial Sequence
<220> <223> Artificial
<220> <221> MISCFEATURE <222> (43)..(43) <223> Xaa is pAF
<400> 4
Ala Pro Leu Ser Pro Ala Ser Ser Leu Pro Gin Ser Phe Leu Leu Lys 1 5 10 15
Cys Leu Glu Gin Val Arg Lys Ile Gin Ala Asp Gly Ala Glu Leu Gin 20 25 30
Glu Arg Leu Cys Ala Thr His Lys Leu Cys Xaa Pro Gin Glu Leu Val 35 40 45
Leu Leu Gly His Ser Leu Gly Leu Pro Gin Ala Ser Leu Ser Ser Cys 50 55 60
Ser Ser Gln Ala Leu Gln Leu Thr Gly Cys Leu Asn Gln Leu His Gly 65 70 75 80
Gly Leu Val Leu Tyr Gln Gly Leu Leu Gln Ala Leu Ala Gly Ile Ser 85 90 95
Pro Glu Leu Ala Pro Ala Leu Asp Ile Leu Gln Leu Asp Val Thr Asp 100 105 110
Leu Ala Thr Asn Ile Trp Leu Gln Met Glu Asp Leu Arg Met Ala Pro 115 120 125
Ala Ser Leu Pro Thr Gln Gly Thr Val Pro Thr Phe Thr Ser Ala Phe 130 135 140
Gln Arg Arg Ala Gly Gly Val Leu Val Val Ser Gln Leu Gln Ser Phe 145 150 155 160
Leu Glu Leu Ala Tyr Arg Val Leu Arg Tyr Leu Ala Glu Pro 165 170
<210> 5 <211> 528 <212> DNA <213> Artificial Sequence
<220> <223> cDNA (artificial) encodes SEQ ID NO: 1&3
<400> 5 atggcccctc tcagccctgc cagctccctg ccccagagct tcctgctcaa gtgcttagag 60
caagtgagga aaatccaggc tgatggcgcc gagctgcagg agaggctgtg tgccacccac 120
aagctgtgcc acccccagga gctggtgctg ctcgggcact ctctgggcct cccccaggct 180
tccctgagca gctgctccag ccaggccctg cagctgactg gctgcctgaa ccaactgcat 240
ggcggcctcg tcctctacca gggcctcctg caggccctgg cgggcatctc cccagagctg 300
gcccccgccc tggacatact gcagctggat gtcaccgact tagccaccaa catctggctg 360
cagatggaag acctgaggat ggccccggcc tcgcttccca cccagggcac cgtgccgacc 420
ttcacctcgg ccttccagcg ccgggcagga ggggtcctgg ttgtctccca gctgcagagc 480
ttcctggagc tggcgtaccg tgtcctgcgc tacctcgccg agccctga 528
<210> 6 <211> 528 <212> DNA <213> Artificial Sequence
<220> <223> cDNA (artificial) encodes SEQ ID NO: 2&4
<400> 6 atggcccctc tcagccctgc cagctccctg ccccagagct tcctgctcaa gtgcttagag 60 caagtgagga aaatccaggc tgatggcgcc gagctgcagg agaggctgtg tgccacccac 120 aagctgtgct agccccagga gctggtgctg ctcgggcact ctctgggcct cccccaggct 180 tccctgagca gctgctccag ccaggccctg cagctgactg gctgcctgaa ccaactgcat 240 ggcggcctcg tcctctacca gggcctcctg caggccctgg cgggcatctc cccagagctg 300 gcccccgccc tggacatact gcagctggat gtcaccgact tagccaccaa catctggctg 360 cagatggaag acctgaggat ggccccggcc tcgcttccca cccagggcac cgtgccgacc 420 ttcacctcgg ccttccagcg ccgggcagga ggggtcctgg ttgtctccca gctgcagagc 480 ttcctggagc tggcgtaccg tgtcctgcgc tacctcgccg agccctga 528
<210> 7 <211> 175 <212> PRT <213> Artificial Sequence
<220> <223> Artificial
<220> <221> MISCFEATURE <222> (1). (1) <223> Xaa is norleucine or methionine
<220> <221> MISCFEATURE <222> (44)..(44) <223> Xaa is pAF
<220> <221> MISCFEATURE <222> (122)..(122) <223> Xaa is norleucine or methionine
<220> <221> MISCFEATURE <222> (127)..(127) <223> Xaa is norleucine or methionine
<400> 7
Xaa Ala Pro Leu Ser Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu 1 5 10 15
Lys Cys Leu Glu Gln Val Arg Lys Ile Gln Ala Asp Gly Ala Glu Leu 20 25 30
Gln Glu Arg Leu Cys Ala Thr His Lys Leu Cys Xaa Pro Gln Glu Leu 35 40 45
Val Leu Leu Gly His Ser Leu Gly Leu Pro Gln Ala Ser Leu Ser Ser 50 55 60
Cys Ser Ser Gln Ala Leu Gln Leu Thr Gly Cys Leu Asn Gln Leu His
Gly Gly Leu Val Leu Tyr Gln Gly Leu Leu Gln Ala Leu Ala Gly Ile 85 90 95
Ser Pro Glu Leu Ala Pro Ala Leu Asp Ile Leu Gln Leu Asp Val Thr 100 105 110
Asp Leu Ala Thr Asn Ile Trp Leu Gln Xaa Glu Asp Leu Arg Xaa Ala 115 120 125
Pro Ala Ser Leu Pro Thr Gln Gly Thr Val Pro Thr Phe Thr Ser Ala 130 135 140
Phe Gln Arg Arg Ala Gly Gly Val Leu Val Val Ser Gln Leu Gln Ser 145 150 155 160
Phe Leu Glu Leu Ala Tyr Arg Val Leu Arg Tyr Leu Ala Glu Pro 165 170 175
<210> 8 <211> 173 <212> PRT <213> Artificial Sequence
<220> <223> Artificial
<220> <221> MISCFEATURE <222> (42)..(42) <223> Xaa is pAF
<400> 8
Pro Leu Ser Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys Cys 1 5 10 15
Leu Glu Gln Val Arg Lys Ile Gln Ala Asp Gly Ala Glu Leu Gln Glu 20 25 30
Arg Leu Cys Ala Thr His Lys Leu Cys Xaa Pro Gln Glu Leu Val Leu 35 40 45
Leu Gly His Ser Leu Gly Leu Pro Gln Ala Ser Leu Ser Ser Cys Ser 50 55 60
Ser Gln Ala Leu Gln Leu Thr Gly Cys Leu Asn Gln Leu His Gly Gly 65 70 75 80
Leu Val Leu Tyr Gln Gly Leu Leu Gln Ala Leu Ala Gly Ile Ser Pro 85 90 95
Glu Leu Ala Pro Ala Leu Asp Ile Leu Gln Leu Asp Val Thr Asp Leu 100 105 110
Ala Thr Asn Ile Trp Leu Gln Met Glu Asp Leu Arg Met Ala Pro Ala 115 120 125
Ser Leu Pro Thr Gln Gly Thr Val Pro Thr Phe Thr Ser Ala Phe Gln 130 135 140
Arg Arg Ala Gly Gly Val Leu Val Val Ser Gln Leu Gln Ser Phe Leu 145 150 155 160
Glu Leu Ala Tyr Arg Val Leu Arg Tyr Leu Ala Glu Pro 165 170
<210> 9 <211> 175 <212> PRT <213> Artificial Sequence
<220> <223> Artificial
<220> <221> MISCFEATURE <222> (44)..(44) <223> Xaa is pAF
<400> 9
Met Ala Pro Leu Ser Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu 1 5 10 15
Lys Ser Leu Glu Gln Val Arg Lys Ile Gln Ala Asp Gly Ala Glu Leu 20 25 30
Gln Glu Arg Leu Cys Ala Thr His Lys Leu Cys Xaa Pro Gln Glu Leu 35 40 45
Val Leu Leu Gly His Ser Leu Gly Leu Pro Gln Ala Ser Leu Ser Ser 50 55 60
Cys Ser Ser Gln Ala Leu Gln Leu Thr Gly Cys Leu Asn Gln Leu His 65 70 75 80
Gly Gly Leu Val Leu Tyr Gln Gly Leu Leu Gln Ala Leu Ala Gly Ile 85 90 95
Ser Pro Glu Leu Ala Pro Ala Leu Asp Ile Leu Gln Leu Asp Val Thr 100 105 110
Asp Leu Ala Thr Asn Ile Trp Leu Gln Met Glu Asp Leu Arg Met Ala 115 120 125
Pro Ala Ser Leu Pro Thr Gln Gly Thr Val Pro Thr Phe Thr Ser Ala 130 135 140
Phe Gln Arg Arg Ala Gly Gly Val Leu Val Val Ser Gln Leu Gln Ser 145 150 155 160
Phe Leu Glu Leu Ala Tyr Arg Val Leu Arg Tyr Leu Ala Glu Pro 165 170 175
<210> 10 <211> 175 <212> PRT <213> Artificial Sequence
<220> <223> Artificial
<220> <221> MISCFEATURE <222> (1)..(1) <223> Xaa is norleucine or methionine
<220> <221> MISCFEATURE <222> (44)..(44) <223> Xaa is pAF
<220> <221> MISCFEATURE <222> (122)..(122) <223> Xaa is norleucine or methionine
<220> <221> MISCFEATURE <222> (127)..(127) <223> Xaa is norleucine or methionine
<400> 10
Xaa Ala Pro Leu Ser Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu 1 5 10 15
Lys Ser Leu Glu Gln Val Arg Lys Ile Gln Ala Asp Gly Ala Glu Leu 20 25 30
Gln Glu Arg Leu Cys Ala Thr His Lys Leu Cys Xaa Pro Gln Glu Leu 35 40 45
Val Leu Leu Gly His Ser Leu Gly Leu Pro Gln Ala Ser Leu Ser Ser 50 55 60
Cys Ser Ser Gln Ala Leu Gln Leu Thr Gly Cys Leu Asn Gln Leu His 65 70 75 80
Gly Gly Leu Val Leu Tyr Gln Gly Leu Leu Gln Ala Leu Ala Gly Ile 85 90 95
Ser Pro Glu Leu Ala Pro Ala Leu Asp Ile Leu Gln Leu Asp Val Thr 100 105 110
Asp Leu Ala Thr Asn Ile Trp Leu Gln Xaa Glu Asp Leu Arg Xaa Ala 115 120 125
Pro Ala Ser Leu Pro Thr Gln Gly Thr Val Pro Thr Phe Thr Ser Ala 130 135 140
Phe Gln Arg Arg Ala Gly Gly Val Leu Val Val Ser Gln Leu Gln Ser 145 150 155 160
Phe Leu Glu Leu Ala Tyr Arg Val Leu Arg Tyr Leu Ala Glu Pro 165 170 175
<210> 11 <211> 174 <212> PRT <213> Artificial Sequence
<220> <223> Artificial
<220> <221> MISCFEATURE <222> (43)..(43) <223> Xaa is pAF
<400> 11
Ala Pro Leu Ser Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys 1 5 10 15
Cys Leu Glu Gln Val Arg Lys Ile Gln Ala Asp Gly Ala Glu Leu Gln 20 25 30
Glu Arg Leu Cys Ala Thr His Lys Leu Cys Xaa Pro Gln Glu Leu Val 35 40 45
Leu Leu Gly His Ser Leu Gly Leu Pro Gln Ala Ser Leu Ser Ser Cys 50 55 60
Ser Ser Gln Ala Leu Gln Leu Thr Gly Cys Leu Asn Gln Leu His Gly 65 70 75 80
Gly Leu Val Leu Tyr Gln Gly Leu Leu Gln Ala Leu Ala Gly Ile Ser 85 90 95
Pro Glu Leu Ala Pro Ala Leu Asp Ile Leu Gln Leu Asp Val Thr Asp 100 105 110
Leu Ala Thr Asn Ile Trp Leu Gln Met Glu Asp Leu Arg Met Ala Pro 115 120 125
Ala Ser Leu Pro Thr Gln Gly Thr Val Pro Thr Phe Thr Ser Ala Phe 130 135 140
Gln Arg Arg Ala Gly Gly Val Leu Val Val Ser Gln Leu Gln Ser Phe 145 150 155 160
Leu Glu Leu Ala Tyr Arg Val Leu Arg Tyr Leu Ala Glu Pro 165 170
<210> 12 <211> 173 <212> PRT <213> Artificial Sequence
<220> <223> Artificial
<220> <221> MISCFEATURE <222> (42)..(42) <223> Xaa is pAF
<400> 12
Pro Leu Ser Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys Ser 1 5 10 15
Leu Glu Gln Val Arg Lys Ile Gln Ala Asp Gly Ala Glu Leu Gln Glu 20 25 30
Arg Leu Cys Ala Thr His Lys Leu Cys Xaa Pro Gln Glu Leu Val Leu 35 40 45
Leu Gly His Ser Leu Gly Leu Pro Gln Ala Ser Leu Ser Ser Cys Ser 50 55 60
Ser Gln Ala Leu Gln Leu Thr Gly Cys Leu Asn Gln Leu His Gly Gly 65 70 75 80
Leu Val Leu Tyr Gln Gly Leu Leu Gln Ala Leu Ala Gly Ile Ser Pro 85 90 95
Glu Leu Ala Pro Ala Leu Asp Ile Leu Gln Leu Asp Val Thr Asp Leu 100 105 110
Ala Thr Asn Ile Trp Leu Gln Met Glu Asp Leu Arg Met Ala Pro Ala 115 120 125
Ser Leu Pro Thr Gln Gly Thr Val Pro Thr Phe Thr Ser Ala Phe Gln 130 135 140
Arg Arg Ala Gly Gly Val Leu Val Val Ser Gln Leu Gln Ser Phe Leu 145 150 155 160
Glu Leu Ala Tyr Arg Val Leu Arg Tyr Leu Ala Glu Pro 165 170
<210> 13 <211> 174 <212> PRT <213> Artificial Sequence
<220> <223> Artificial
<220> <221> MISCFEATURE <222> (1). (1) <223> Xaa is methionine-alanine, norleucine-alanine, alanine, or no amino acid
<220> <221> MISCFEATURE <222> (17)..(17) <223> Xaa is cysteine or serine <220> <221> MISCFEATURE <222> (43)..(43) <223> Xaa is pAF
<220> <221> MISCFEATURE <222> (121)..(121) <223> Xaa is methionine or norleucine
<220> <221> MISCFEATURE <222> (126)..(126) <223> Xaa is methionine or norleucine
<400> 13
Xaa Pro Leu Ser Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys 1 5 10 15
Xaa Leu Glu Gln Val Arg Lys Ile Gln Ala Asp Gly Ala Glu Leu Gln 20 25 30
Glu Arg Leu Cys Ala Thr His Lys Leu Cys Xaa Pro Gln Glu Leu Val 35 40 45
Leu Leu Gly His Ser Leu Gly Leu Pro Gln Ala Ser Leu Ser Ser Cys 50 55 60
Ser Ser Gln Ala Leu Gln Leu Thr Gly Cys Leu Asn Gln Leu His Gly 65 70 75 80
Gly Leu Val Leu Tyr Gln Gly Leu Leu Gln Ala Leu Ala Gly Ile Ser 85 90 95
Pro Glu Leu Ala Pro Ala Leu Asp Ile Leu Gln Leu Asp Val Thr Asp 100 105 110
Leu Ala Thr Asn Ile Trp Leu Gln Xaa Glu Asp Leu Arg Xaa Ala Pro 115 120 125
Ala Ser Leu Pro Thr Gln Gly Thr Val Pro Thr Phe Thr Ser Ala Phe 130 135 140
Gln Arg Arg Ala Gly Gly Val Leu Val Val Ser Gln Leu Gln Ser Phe 145 150 155 160
Leu Glu Leu Ala Tyr Arg Val Leu Arg Tyr Leu Ala Glu Pro 165 170
EDITORIAL NOTE
202104727
Page numbers 24 through to 36 are located as the gene sequence

Claims (40)

WHAT IS CLAIMED IS:
1. A porcine granulocyte colony stimulating factor (pG-CSF) variant consisting of a sequence of: X 1PLSPASSLPQSFLLKX 2LEQVRKIQADGAELQERLCATHKLC(pAF)PQELVLLGHSL GLPQASLSSCSSQALQLTGCLNQLHGGLVLYQGLLQALAGISPELAPALDILQLDVTD LATNIWLQX 3EDLRX 3APASLPTQGTVPTFTSAFQRRAGGVLVVSQLQSFLELAYRVL RYLAEP (SEQ ID NO: 13); wherein X 1 is selected from the group of methionine alanine, norleucine alanine, alanine only, and no amino acids); wherein X 2 is cysteine or serine; wherein X 3 is methionine or norleucine; and wherein a para-acetyl phenylalanine (pAF) synthetic amino acid present at position 43 is covalently attached to a poly(ethylene glycol) (PEG).
2. The pG-CSF variant of Claim 1, wherein the PEG has a molecular weight of about 20 kD to about 50 kD.
3. The pG-CSF variant of Claim 1 or 2, wherein the PEG has a molecular weight of about 30 kD.
4. The pG-CSF variant of any one of Claims 1-3, wherein the PEG is linear.
5. A porcine granulocyte colony stimulating factor (pG-CSF) variant consisting of an amino acid sequence of: MAPLSPASSLPQSFLLKCLEQVRKIQADGAELQERLCATHKLC(pAF)PQELVLLGHSL GLPQASLSSCSSQALQLTGCLNQLHGGLVLYQGLLQALAGISPEL APALDILQLDVTDLATNIWLQMEDLRMAPASLPTQGTVPTFTSAFQRRAG GVLVVSQLQSFLELAYRVLRYLAEP (SEQ ID NO: 2), wherein a para-acetyl phenylalanine (pAF) synthetic amino acid present at position 43 is covalently attached to a 30 kD linear PEG.
6. A pharmaceutical composition comprising the pG-CSF variant of any of Claims 1 to 5, and at least one pharmaceutically acceptable carrier, diluent, or excipient.
7. A method for treating a bacterial infection in a porcine comprising administering a therapeutically effective amount of the pG-CSF variant of Claims 1 to 5 or the pharmaceutical composition of Claim 6 to the porcine in need thereof.
8. The method of Claim 7, wherein the bacterial infection is mastitis, metritis and agalactia (MMA) syndrome.
9. The method of Claims 7 or 8, wherein the porcine is a periparturient sow.
10. The method of any of Claims 7 - 9, wherein the therapeutically effective amount of pG-CSF is about 10 - 100 jg/kg animal weight.
11. The method of any of Claims 7 - 9, wherein the therapeutically effective amount of pG-CSF is about 30 - 50 pg/kg animal weight.
12. The method of any of Claims 7 - 9, wherein the therapeutically effective amount of pG-CSF is about 40 pg/kg animal weight.
13. The method of any one of Claims 7 - 12, wherein the administering occurs at least once within 7 days prior to farrowing.
14. The method of any one of Claims 7 - 13, wherein the administering occurs at farrowing.
15. The method of any one of Claims 7 - 14, further comprising a second administration no later than 14 days after farrowing.
16. A method for reducing piglet mortality comprising administering a therapeutically effective amount of the pG-CSF variant of Claims 1 to 5 or the pharmaceutical composition of Claim 6 to a periparturient sow.
17. The method of Claim 16, wherein the therapeutically effective amount of pG-CSF is about 10 - 100 jg/kg animal weight.
18. The method of Claim 16, wherein the therapeutically effective amount of pG-CSF is about 30 - 50 pg/kg animal weight.
19. The method of Claim 16, wherein the therapeutically effective amount of pG-CSF is about 40 pg/kg animal weight.
20. The method of any one of Claims 16 - 19, wherein the administering occurs at least once within 7 days prior to farrowing.
21. The method of any one of Claims 16 - 20, wherein the administering occurs at farrowing.
22. The method of any one of Claims 16 - 21, further comprising a second administration no later than 14 days after farrowing.
23. A method for stimulating innate immune response in a porcine comprising administering a therapeutically effective amount of the pG-CSF variant of Claims 1 to 5 or the pharmaceutical composition of Claim 6 to the porcine in need thereof.
24. The method of Claim 7, wherein the innate immune response comprises increasing neutrophil antibacterial function, cytokine levels, activating or increasing levels of dendritic cells, activating or increasing levels of lymphocytes, or activating or increasing levels of myelocytes, or any combination thereof.
25. The method of Claims 23 or 24, wherein the porcine is a periparturient sow.
26. The method of any of Claims 23 - 25, wherein the therapeutically effective amount of pG-CSF is about 10 - 100 pg/kg animal weight.
27. The method of any of Claims 23 - 25, wherein the therapeutically effective amount of pG-CSF is about 30 - 50 pg/kg animal weight.
28. The method of any of Claims 23 - 25, wherein the therapeutically effective amount of pG-CSF is about 40 pg/kg animal weight.
29. The method of any one of Claims 23 - 28, wherein the administering occurs at least once within 7 days prior to farrowing.
30. The method of any one of Claims 23 - 29, wherein the administering occurs at farrowing.
31. The method of any one of Claims 23 - 30, further comprising a second administration no later than 14 days after farrowing.
32. A pG-CSF variant of any one of Claims 1 to 5 for use in therapy.
33. A pG-CSF variant of any one of Claims I to 5 for use in the treatment of a bacterial infection in a porcine.
34. A pG-CSF variant of any one of Claims 1 to 5 for use in the treatment of a bacterial infection in a periparturient sow.
35. A pG-CSF variant of any one of Claims I to 5 for use in increasing neutrophil levels in a periparturient sow.
36. A pG-CSF variant of any one of Claims 1 to 5 for use in reducing piglet mortality, wherein the pG-CSF variant is administered to a periparturient sow to treat a bacterial infection.
37. The pG-CSF variant for use according to any of Claims 32 - 36, wherein the pG-CSF variant is administered at about 10 - 100 pg/kg animal weight, about 30 - 50 pg/kg animal weight, or about 40 pg/kg animal weight.
38. The pG-CSF variant for use according to any of Claims 32 - 37, wherein the pG-CSF variant is administered at least once within 7 days prior to farrowing.
39. The pG-CSF variant for use according to any of Claims 32 - 37, wherein the pG-CSF variant is administered at farrowing.
40. The pG-CSF variant for use according to any of Claims 32 - 39, wherein a second dose of a pG-CSF variant is administered no later than 14 days after farrowing.
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Family Cites Families (36)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4810643A (en) * 1985-08-23 1989-03-07 Kirin- Amgen Inc. Production of pluripotent granulocyte colony-stimulating factor
US4961926A (en) * 1987-11-19 1990-10-09 Sloan-Kettering Institute For Cancer Research Methods for prevention and treatment of mucositis with granulocyte colony stimulating factor
WO1989010932A1 (en) * 1988-05-13 1989-11-16 Amgen Inc. Compositions and method for treating or preventing infections in animals
JPH04502164A (en) * 1989-10-10 1992-04-16 アムジエン・インコーポレーテツド Chemical compositions and methods for the treatment or prevention of infections in canines and felines
AU3515001A (en) * 1993-01-28 2001-06-28 Amgen, Inc. G-CSF analog compositions
CA2139385C (en) * 1994-02-04 2001-12-25 Gottfried Alber Products containing g-csf and tnf binding protein
RU2273645C9 (en) * 1994-08-17 2006-11-27 Дзе Рокефеллер Юниверсити Obesity polypeptide (ob) (variants), its analogue (variants), and fused protein (variants), nucleic acid isolated molecule, dna molecule, cloning recombinant vector, expression recombinant vector, pharmaceutical composition, monoclonal and polyclonal antibody
CA2452540A1 (en) * 1996-10-15 1998-04-23 Amgen Inc. Keratinocyte growth factor-2 products
JP2004511201A (en) * 1998-10-09 2004-04-15 カイロン　コーポレイション Neisseria genome sequences and methods of using them
JP2002540065A (en) 1999-01-29 2002-11-26 アムジェン インコーポレーテッド GCSF conjugate
US7323174B1 (en) * 2000-06-12 2008-01-29 Arizona Board Of Regents On Behalf Of The University Of Arizona Modulation of immune response and methods based thereon
MXPA04000068A (en) * 2001-06-22 2005-06-06 Pharmacia Corp Chemically-modified progenipoietin conjugates.
US7173003B2 (en) * 2001-10-10 2007-02-06 Neose Technologies, Inc. Granulocyte colony stimulating factor: remodeling and glycoconjugation of G-CSF
BR0213402A (en) * 2001-10-29 2004-10-13 Crucell Holland Bv Methods for identifying a mammalian cell capable of producing a proteinaceous molecule, for selecting a mammalian cell capable of producing a proteinaceous molecule, for obtaining a mammalian cell from a heterogeneous population of cells, and for producing a proteinaceous molecule, pharmaceutically acceptable composition, recombinantly produced erythropoietin, uses of a mammalian cell, recombinantly produced erythropoietin, and an erythropoietin-like molecule composition, pharmaceutical preparation, method for the preventive and / or therapeutic treatment of a disorder, similar molecule composition to erythropoietin, methods for producing proteinaceous molecules in a mammalian cell, to produce a fraction enriched in a proteinaceous molecule, and to fractionate a mixture containing proteinaceous molecules, fraction, and, uses of a fraction or composition, and erythropoietin recombinantly produced in a mammalian cell
CN1241946C (en) * 2002-07-01 2006-02-15 美国福源集团 Human serum albumins recombined merge protein having hyperplasia stimulation function to multiple cells
US7785601B2 (en) 2002-12-31 2010-08-31 Sygnis Bioscience Gmbh & Co. Kg Methods of treating neurological conditions with hematopoietic growth factors
CN100522988C (en) * 2003-04-09 2009-08-05 诺和诺德公司 Glycopegylation methods and proteins/peptides produced by the methods
NZ543392A (en) 2003-05-09 2009-02-28 Schering Ag Treatment of inflammatory respiratory diseases e.g. SARS, ARDS, IRDS
MXPA06008496A (en) 2004-02-02 2007-01-30 Ambrx Inc Modified human interferon polypeptides and their uses.
CN101014616A (en) * 2004-07-26 2007-08-08 埃斯特瑞恩有限公司 Linkers
KR100694994B1 (en) * 2005-06-13 2007-03-14 씨제이 주식회사 Human Granulocyte-Colony Stimulating Factor Isoforms
KR100735784B1 (en) * 2005-07-20 2007-07-06 재단법인 목암생명공학연구소 Mutant of granulocyte-colony stimulating factorG-CSF and chemically conjugated polypeptide thereof
EP1834962A1 (en) * 2006-03-15 2007-09-19 Biotecon Therapeutics GmbH PEGylated mtutated Clostridium botulinum toxin
CN101245109B (en) * 2007-02-12 2011-12-14 杭州九源基因工程有限公司 Polyglycol single-modificatory recombined human granular leukocyte colony stimulating factor mutant and preparation method thereof
JP4788646B2 (en) * 2007-04-26 2011-10-05 トヨタ自動車株式会社 Power storage device and vehicle
CN101352573B (en) * 2007-07-27 2011-02-09 杭州九源基因工程有限公司 Recombinant human granulocyte colony stimulating factor lysine defect body modified by polyethyleneglycol
EP2185583A2 (en) * 2007-08-03 2010-05-19 Asterion Limited Granulocyte colony stimulating factor
AU2013202836B8 (en) 2008-07-23 2015-07-30 Ambrx, Inc. Modified bovine G-CSF polypeptides and their uses
US10138283B2 (en) 2008-07-23 2018-11-27 Ambrx, Inc. Modified bovine G-CSF polypeptides and their uses
JP2012518014A (en) * 2009-02-17 2012-08-09 シグニス・バイオサイエンス・ゲゼルシャフト・ミット・ベシュレンクテル・ハフツング・ウント・コンパニー・コマンディートゲゼルシャフト Use of G-CSF to extend the therapeutic time of thrombolytic therapy for stroke
RU2519031C1 (en) * 2010-01-19 2014-06-10 Ханми Сайенс Ко., Лтд. Liquid formulations for long-acting conjugate of g-csf
WO2011163460A1 (en) * 2010-06-24 2011-12-29 Indiana University Research And Technology Corporation Yl-based insulin-like growth factors exhibiting high activity at the insulin receptor
AR083006A1 (en) * 2010-09-23 2013-01-23 Lilly Co Eli FORMULATIONS FOR THE STIMULATING FACTOR OF COLONIES OF GRANULOCITS (G-CSF) BOVINE AND VARIANTS OF THE SAME
BR112013031943B1 (en) * 2011-06-13 2021-10-13 Csl Limited COMPOSITION INCLUDING PROTEINS AND ANTIBODIES AGAINST G-CSFR
BR112014015156A2 (en) * 2011-12-20 2020-10-27 Indiana University Research And Technology Corporation ctp-based insulin analogues, their methods of production and use in the treatment of hyperglycemia, as well as nucleic acid and host cell sequences
US9605040B2 (en) * 2012-03-26 2017-03-28 Axcella Health Inc. Nutritive proteins and methods

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